Studies on the endogenous L-selectin ligands: systematic and highly efficient total synthetic routes to lactamized-sialyl 6-O-sulfo Lewis X and other novel gangliosides containing lactamized neuraminic acid

Systematic syntheses of lactamized neuraminic acid-containing gangliosides GM4, sulfated sialylparagloboside, and sulfated/nonsulfated sialyl Lewis X are described. The highly efficient, one-step lactamization of neuraminic acid was accomplished by treatment of the N-deacetylated sialic acid (neuraminic acid)-containing gangliosides with HBTU and HOBt in DMF at 65 degrees C. Both the lactamized neuraminic acid residue and the sulfate group at O-6 of the GlcNAc residue were found to be involved in the antigenic determinant defined by G159 monoclonal antibody, while the fucose residue may not be critical for the recognition by G159 mAb.     

A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.     

ecNOS gene polymorphism is associated with endothelium-dependent vasodilation in Type 2 diabetes

The association between endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphism and vascular endothelial function has not been clarified. We investigated the impact of ecNOS gene polymorphism on endothelial function in 95 patients with Type 2 diabetes (ecNOS genotype: 4b/b, n = 62; 4b/a, n = 30; 4a/a, n = 3). Flow-mediated (endothelium dependent, FMD) and nitroglycerin-induced (endothelium independent, NTG) vasodilations of the right brachial artery were studied using a phase-locked echotracking system. There were no significant differences in clinical characteristics among the ecNOS genotypes. The FMD was significantly lower in the patients with ecNOS4a allele than in those without ecNOS4a allele (P < 0.05). Multiple regression analysis showed that ecNOS4a allele and mean blood pressure were significant independent determinants for reduced FMD in all patients (R(2) = 0.122, P = 0.0025). The ecNOS4a allele was an independent determinant for reduced FMD in smokers but not in nonsmokers. These results suggest that ecNOS4a allele is a genetic risk factor for endothelial dysfunction in diabetic patients, especially in smokers.     

A molecular marker diagnostic of a specific isolate of an arbuscular mycorrhizal fungus,Gigaspora margarita

To investigate the auto-ecology of a strain of Gigaspora margarita in a commercial inoculum, we found a pair of PCR primers amplifying a sequence of 235 bp diagnostic of the isolate. We designed an oligonucleotide probe based on the DNA sequence. The combination of PCR and the probing successfully detected the diagnostic sequence from both DNA preparations of single spores and colonized roots. This protocol enabled us to distinguish the isolate among several isolates from Japan, Nepal and the USA.     

Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.     

Acid sphingomyelinase activation requires caspase-8 but not p53 nor reactive oxygen species during Fas-induced apoptosis in human glioma cells

During apoptosis of human glioma cells induced by anti-Fas antibody, ceramide formation with activation of acid, but not neutral sphingomyelinase (SMase), was observed. A potent inhibitor of acid SMase, SR33557, effectively inhibited ceramide formation and apoptosis. Fas-induced apoptosis and ceramide formation proceeded regardless of p53 status. The agents, which modify intracellular levels of reactive oxygen species (ROS) and reduced glutathione (GSH), failed to modulate Fas-induced acid SMase activation and apoptosis. Moreover, expression of functional p53 protein using a temperature-sensitive human p53val(138) induced ceramide generation by activation of neutral SMase but not acid SMase through ROS formation. Peptide inhibitors for caspases-8 (z-IETD-fmk) and -3 (z-DEVD-fmk) suppressed Fas-induced apoptosis. However, activation of acid SMase was inhibited only by z-IETD-fmk. Thus, ceramide generated by acid SMase may take a part in Fas-induced apoptosis of human glioma cells and acid SMase activation may be dependent on caspase-8 activation, but not on p53 nor ROS.     

The leukocyte common antigen (CD45) of the Pacific hagfish,Eptatretus stoutii: implications for the primordial function of CD45

CD45, originally known as the leukocyte common antigen, is a prototypical transmembrane protein tyrosine phosphatase that plays a critical role in signal transduction through T-cell and B-cell receptors, as well as in T-cell and B-cell development. In the present study, we show that the Pacific hagfish, widely believed to lack the adaptive immune system, has CD45. The presence of CD45 in jawless fish is consistent with the recent discovery that CD45 also plays a crucial role in innate immunity via the regulation of signaling through type I and type II cytokine receptors. It is likely that CD45 was recruited to activate lymphocytes through antigen receptors encoded by rearranging genes in jawed vertebrates.     

EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)-hydroxyarginine

Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 degrees C the oxyferrous-NOS-substrate complexes of both L-arginine (Arg) and N(G)-hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O2 to the ferroheme of NOS recruits H2O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis.     

Two novel oligosaccharides formed by 1F-fructosyltransferase purified from roots of asparagus (Asparagus officinalis L.)

Two novel oligosaccharides, tetra-and penta-saccharides were synthesized by fructosyl transfer from 1-kestose to 4G-beta-D-galactopyranosylsucrose with a purified 1F-fructosyltransferase of asparagus roots and identified as 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose, O-beta-D-fructofuranosyl-(2-->1)-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside and 1F(1-beta-D-fructofuranosyl)2-4G-beta-D-galactopyranosylsucrose, [O-beta-D-fructofuranosyl-(2-->1)]2-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside, respectively. Both oligosaccharides were scarcely hydrolyzed by carbohydrase from rat small intestine. Human intestinal bacterial growth by 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose was compared with that by the tetrasaccharides, stachyose and nystose. Bifidobacteria utilized 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose to the same extent as stachyose or nystose. On the other hand, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcusfaecalis, that produce mutagenic substances did not use the synthetic oligosaccharide.     

Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.